Efficient repair of spontaneous and induced DNA lesions is critical for maintaining the mitotic stability of eukaryotic genomes. Double-strand breaks (DSBs) are an especially toxic lesion and are repaired by two distinct pathways: homologous recombination and non-homologous end joining (HR and NHEJ, respectively). The NHEJ pathway ligates ends that often require processing, which results in loss/gain of sequence at the joint and renders the process highly error-prone. By contrast, HR restores a broken molecule by copying information from an intact donor, and thus is considered a high-fidelity process. Even so, HR can alter the linkage of sequences that flank the repair tract to result in loss of heterozygosity, or engage dispersed repeated sequences to generate chromosome rearrangements. Both HR and NHEJ are essential in mammals and defects have been linked to a large number of human diseases that include neurological disorders, immune system dysfunction, premature aging syndromes and cancer. Neither HR nor NHEJ is essential in lower eukaryotes, however, allowing the underlying mechanisms and their genetic control to be studied. The proposed experiments will use budding yeast as a model genetic system to elucidate molecular intermediates and genetic mechanisms of HR and NHEJ. Aims 1 and 2 will employ nucleases that create single, defined DSBs in the yeast genome and will monitor repair using novel, selective systems. In Aim 1, enzymes that generate different types of broken ends will be used to determine how end structure/sequence affects the NHEJ process and alters genetic outcome. Using a robust method developed to monitor DNA strand transfer during HR, Aim 2 will identify mechanisms that confer distinctive asymmetries on the final recombination products. Finally, Aim 3 will define strand-exchange intermediates associated with spontaneous recombination events and thereby resolve a long-standing issue in the HR field: whether spontaneous events most often initiate with a single-strand nick/gap or with a DSB. The high conservation in HR and NHEJ mechanisms makes the proposed yeast studies relevant to issues of genetic stability that impact human health.